Origin and evolution of cultivated Agrostis spp. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at George Mason University By Keenan L. Amundsen Master of Science Michigan State University, 2003 Director: Donald Seto, Associate Professor Department of Bioinformatics and Computational Biology Fall Semester 2009 George Mason University Fairfax, VA Copyright 2009 Keenan L. Amundsen All Rights Reserved ii ACKNOWLEDGEMENTS I would like to thank my committee members Dr. Don Seto, Dr. Andrea Weeks, Dr. Huzefa Rangwala, and Dr. Scott Warnke for their support and guidance throughout the completion of this project. Thanks are also due to Dr. John Grefenstette, a former committee member, for assistance during the early stages of this study. I would also like to thank the United States Golf Association and Dr. Scott Warnke of the Floral and Nursery Plants Research Unit, United States Department of Agriculture for providing financial support toward academic and research expenses. Additional thanks are extended to Ms. Vicki Bradley of the National Plant Germplasm System, Dr. Doug Johnson of the Forage and Range Research Laboratory, United States Department of Agriculture, Dr. Geunhwa Jung of the University of Massachusetts, Dr. Faith Belanger of Rutgers, the State University of New Jersey, and Mr. Kevin Morris executive director of the National Turfgrass Evaluation Program for providing research materials invaluable to the completion of this research. iii TABLE OF CONTENTS Page List of Tables ....................................................................................................................v List of Figures................................................................................................................. vi Abstract.......................................................................................................................... vii 1. Introduction.................................................................................................................1 2. Ploidy Analysis of NPGS Agrostis Accessions ........................................................23 3. MITE-Display Marker Development in Agrostis .....................................................37 4. MITE-Display Diversity Analysis in Agrostis..........................................................50 5. Evolution of NPGS Agrostis Accessions Based on trnL-trnF and atpI-atpH Intergenic Spacer Regions ........................................................................................65 6. Conclusion ................................................................................................................86 Appendix A List of Germplasm and 2C DNA Content..................................................89 Appendix B MITE-Insertional Polymorphism Primer Pairs ..........................................97 Appendix C Alignment of trnL-trnF Intergenic Spacer Region...................................102 Appendix D Alignment of atpI-atpH Intergenic Spacer Region..................................108 References.....................................................................................................................118 iv LIST OF TABLES Table Page Table 1 Predicted Diploids Based on 2C DNA Content...............................................31 Table 2 Predicted Tetraploids Based on 2C DNA Content ..........................................32 Table 3 Validation of Flow Cytometry Ploidy Predictions...........................................35 Table 4 MITE-Display Primer Sequences ....................................................................43 Table 5 Number of Candidate MITEs from Agrostis DNA Sequence Libraries..........45 Table 6 Number of MITE-Display Markers Generated With Each Primer Pair ..........55 Table 7 MITE-Display Diversity Study Accession List ...............................................56 Table 8 Accessions Used in Phylogenetic Analyses.....................................................69 Table 9 Conserved Chloroplast Primers .......................................................................71 Table 10 Unique trnL-trnF Intergenic Spacer Haplotypes .............................................73 Table 11 Unique atpI-atpH Intergenic Spacer Haplotypes.............................................73 v LIST OF FIGURES Figure Page Figure 1 Ploidy Analyzer Trace Data ...........................................................................29 Figure 2 Feulgen Stained Chromosomes......................................................................33 Figure 3 Sequence alignment of MDM-2 MITEs from Oryza sativa...........................40 Figure 4 Agrostis Diploid MIP Screen .........................................................................46 Figure 5 MITE-Display Test.........................................................................................48 Figure 6 Cluster Analysis of 81 Agrostis Accessions...................................................57 Figure 7 Structure Analysis of 81 Agrostis Accessions................................................59 Figure 8 Principal Component Analysis of 81 Agrostis Accessions ............................60 Figure 9 Phylogenetic Analysis of trnL-trnF Intergenic Spacer ..................................75 Figure 10 Phylogenetic Analysis of atpI-atpH Intergenic Spacer..................................76 Figure 11 Phylogenetic Analysis of trnL-trnF Intergenic Spacer from cultivated Agrostis...........................................................................................................78 Figure 12 Phylogenetic Analysis of atpI-atpH Intergenic Spacer from cultivated Agrostis...........................................................................................................79 Figure 13 Consensus Network of the atpI-atpH and trnL-trnF Spacer Regions............80 Figure 14 Consensus Network of the Intergenic Spacer Regions and MITE-Display Data ................................................................................................................82 vi ABSTRACT ORIGIN AND EVOLUTION OF CULTIVATED AGROSTIS SPP. Keenan L. Amundsen, PhD. George Mason University, 2009 Dissertation Director: Dr. Donald Seto Creeping bentgrass (Agrostis stolonifera L.) is a highly outcrossing allotetraploid species important to the turfgrass industry because it has unique growth and aesthetic characteristics that make it ideally suited for use in high quality turf stands. There are between 150 and 200 species of Agrostis (bentgrasses) and the relationships between species are not clearly understood. Resolving these relationships is complicated in part because many of the species share similar morphological features and interspecific hybridization is common, making taxonomic classification difficult. Knowledge of the evolution of Agrostis species would provide turfgrass breeders with information on the origins of species and potential sources of novel germplasm. In the present study 305 Agrostis accessions were examined to explore the evolutionary relationships within the Agrostis genus. Flow cytometry experiments were performed on each accession to measure DNA content and make ploidy predictions. MITE display molecular markers were designed and 1,309 were used to assess genetic diversity within a subset of these Agrostis accessions. Fragments of the trnL-trnF and atpI-atpH intergenic spacer regions of the chloroplast genome were also sequenced and used to infer the phylogeny of the chloroplast genome. This data allowed for the prediction of candidate diploid progenitors of cultivated Agrostis species, identification of geographic regions of diverse germplasm, and demonstration of narrowing of the cultivated Agrostis gene pool. The data presented here gives Agrostis breeders valuable information to incorporate novel germplasm in their programs and a direction for recreating the polyploidization events that have led to the cultivated turf-type Agrostis species. 1. INTRODUCTION Creeping bentgrass (Agrostis stolonifera L.) is the most widely utilized cool- season turf species for highly managed playing surfaces such as bowling greens and golf course greens, tees, and fairways (Turgeon, 1996). It outperforms other cool season turfgrass species because of its strong stoloniferous growth, ability to maintain a high level of uniformity after mowing, fine leaf texture, high shoot density, and tolerance of mowing down to heights of 3 mm (Warnke, 2003). A. stolonifera is well adapted to periodically flooded, well-drained, fine-textured, fertile soils (Beard, 1973). It is believed to have originated in Eurasia and is found throughout the world in cool, humid temperate climates (Harvey, 2007). While it is one of the most hardy of the cool season grasses used as turf, the quality of A. stolonifera is adversely affected by wear, soil compaction and a number of pathogens such as dollar spot (Sclerotinia homoeocarpa F.T. Benn.), brown patch (Rhizoctonia solani Kühn), and anthracnose (Colletotrichum cereale Manns). There are 26 Agrostis (bentgrasses) species known within the North American region north of Mexico and 150 to 200 species worldwide (Harvey, 2007). The five species predominantly used as turf are A. stolonifera, A. capillaris L. (colonial bentgrass), A. castellana Boiss. and Reut. (dryland bentgrass), A. canina L. (velvet bentgrass), and A. gigantea Roth. (redtop bentgrass) (Warnke, 2003). Species delimitation based on 1 morphological characters is difficult because of similar features and